Project Summary This proposal is centered upon macromolecular signaling complexes involving KCNE family ion channel regulatory (?) subunits. The KCNE subunits are single-pass transmembrane ? subunits known for modifying the functional properties of voltage-gated potassium (Kv) channel ? subunits such as KCNQ1, in tissues including the auditory system and cardiac myocytes. Each of the five human KCNE subunits can regulate multiple different Kv channel ? subunits, typically forming heteromeric complexes with unique functional attributes compared to those of other subunit compositions. In addition, many of the forty known Kv ? subunits in the human genome are known to be regulated by more than one KCNE isoform. Numerous such complexes have been identified and their absolute necessity in mammalian physiology elucidated by functional studies in combination with either human or mouse genetics, or in some cases both. Despite their necessity for crucial biological processes and linkage to debilitating human diseases, and the potential to leverage KCNE subunit influence on pharmacology to increase the specificity and efficacy of channel-targeted drugs, fundamental questions surrounding the stoichiometry, subunit dynamics, and compositional flexibility of KCNE-containing complexes remain unanswered. In addition, we recently discovered that the KCNQ1-KCNE2 potassium channel forms reciprocally regulating complexes with several sodium-coupled solute transporters ? a further, novel class of signaling complexes about which even less is currently understood. To address these major gaps in knowledge, in the proposed project we will employ cutting-edge fluorescence dynamics techniques to enable visualization of channel complex dynamics at the cell surface, and test novel and important hypotheses that have been suggested by investigators in the field, but not directly tested. In Aim 1 we will employ TIRF, image Mean Square Displacement (iMSD) and Number and Brightness analysis to test the longstanding hypothesis that KCNQ1 channels can lose or gain KCNE subunits at the cell surface, and also elucidate subunit stoichiometry for a variety of KCNE-containing potassium channel complexes, including those formed with solute transporters. In Aim 2, we will use TIRF, confocal microscopy, cross-correlation raster-scan image correlation spectroscopy (ccRICS) and iMSD to elucidate whether KCNQ1 complexes can contain more than one KCNE isoform at a time, and whether these new KCNE subunits can join existing KCNE subunits in complexes with KCNQ1 at the cell surface. Harnessing and developing new approaches to answer longstanding questions about the dynamic capabilities of KCNE-based channels will deliver unprecedented information about this widespread class of ion channels crucial to the healthy functioning of auditory, cardiac, and other tissues. In addition, optimization of these approaches to tackle Kv-KCNE complexes will also open up these techniques to answer similar questions for other ion channels and multi-subunit membrane proteins in general.